Microfabricated two-pin liquid sample dispensing system

ABSTRACT

A two-pin liquid sample dispensing system is provided. The two-pin dispensing system comprises a pair of separately movable pins for holding a droplet of liquid therebetween. Each pin includes a tip spaced predetermined distance from the other pin to define a sample acquisition region. The pins acquire and hold a droplet of the liquid sample in the sample acquisition region formed in the space between the tips and apply the droplet to a selected sample handing system. The distance between the tips is variable to accommodate different liquid samples having varying physical properties and to vary the volume of the acquired droplet.

REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 10/027,171,filed Dec. 21, 2001, which claims priority to U.S. Provisional PatentApplication No. 60/325,001 filed Sep. 25, 2001 and is related to U.S.patent application Ser. No. 10/028,852, entitled “Microfluidic SystemIncluding a Virtual Wall Fluid Interface Port for Interfacing Fluidswith the Microfluidic System”, U.S. patent application Ser. No.10/027,484, entitled “Microfluidic System Including a Virtual Wall FluidInterface Port for Interfacing Fluids with the Microfluidic System”,U.S. patent application Ser. No. 10/027,516, entitled “MicrofluidicSystem Including a Virtual Wall Fluid Interface Port for InterfacingFluids with the Microfluidic System”, U.S. patent application Ser. No.10/027,922, entitled “Small Molecule Substrate Based Enzyme ActivityAssays”, and U.S. patent application Ser. No. 10/029,108 entitled“Droplet Dispensing System”. The contents of the foregoing patentapplications are herein incorporated by reference. The contents of allreferences, issued patents, or published patent applications citedherein are expressly incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a liquid dispensing system for formingand dispensing droplets of a liquid sample.

BACKGROUND OF THE INVENTION

Many chemical, biomedical, bioscience and pharmaceutical industriesrequire chemical operations, such as reactions, separations andsubsequent detection steps, to be performed on samples. It is generallydesirable to introduce these samples into a sample handling system, suchas a microfluidic system capable of handling and analyzing chemical andbiological specimens, quickly, efficiently and in a highly controllablemanner.

Many methods have been described for the interfacing of fluids, e.g.,samples, analytes, reagents, precursors for synthesis and buffers,towards, within or between microfluidic systems. Generally, introductionof a liquid sample to a microfluidic system is accomplished throughsample channels or sample wells. To introduce a liquid sample to themicrofluidic system, a capillary tube may be provided, which dispenses aliquid sample to a sample well, sample channel or other sampleintroduction port. A significant drawback of using a capillary tubeconcerns the low injection efficiency inherent to capillary tubes, thatis, the ratio between the volume of liquid required for a particularchemical operation in a part of the microfluidic system, and the totalvolume of liquid required for the introductory operation. Moreover, itis generally difficult to control the precise volume of dispensed sampleusing capillary tubes. Furthermore, capillary tubes are subject tocontamination, because the same port used to fill the tube is also usedto eject the liquid sample.

U.S. Pat. No. 6,101,946 of Martinsky, the contents of which are hereinincorporated by reference, describes a pin-based system for printingmicroarrays of biochemical substances. The microarray printing systemcomprises a stainless steel printing pin having a sample channel and aflat tip that is machined with an electronic discharge machine (EDM).The pin applies a biochemical substance by filling the sample channeland subsequently directly contacting a printing substrate, to deliverthe sample from the sample channel to the printing substrate. A drawbackof the pin-based system described in the '946 patent concerns theability to control the amount of delivered sample. The pin-based systemis subject to contamination and breakage, because it requires directcontact between the pin tip and the printing substrate. Another drawbackconcerns the difficulty of precisely positioning the tip of the pin toprovide sufficient contact to result in delivery of a sample.

U.S. Pat. No. 6,110,426 of Shalon et al., the contents of which areherein incorporated by reference, describes a capillary dispenser forforming microarrays of biological samples. The capillary dispensercomprises an elongate open capillary channel adapted to hold a liquidsample. The channel is formed by a pair of spaced-apart, coextensive,elongate members, which are tapered toward one another and converge at atip region at the lower end of the channel. The elongate members arefixed relative to each other and the capillary channel is limited to afixed volume. Furthermore, it is difficult to control the amount ofsample that is acquired and dispensed from the capillary dispenser ofthe '246 patent.

SUMMARY OF THE INVENTION

The present invention provides a sample dispensing system comprising twomicrofabricated interacting pins for forming and dispensing droplets ofa liquid sample. Each pin includes a tip spaced predetermined distancefrom the other pin to define a sample acquisition region. The pinsacquire and hold a droplet of the liquid sample in the sampleacquisition region formed in the space between the tips and apply thedroplet to a selected sample handing system. The distance between thetips is variable to accommodate different liquid samples having varyingphysical properties and to vary the volume of the acquired droplet.

According to a first aspect, a droplet dispensing system is provided.The droplet dispensing system comprises two separately movable pins forholding a droplet of a liquid sample. The droplet dispensing systemcomprises a holder, a first pin connected to the holder and having afirst tip and a second pin connected to the holder and having a secondtip spaced an initial separation distance from the first tip to form asample acquisition region for holding a predetermined volume of liquidsample. The initial separation distance is variable.

According to a second aspect, a method of applying a droplet of liquidsample to a substrate is provided. The method comprises providing adispensing system comprising two pins separated by a variable distance,immersing the pin tips in a reservoir to acquire a droplet of a liquidsample, and contacting the substrate to deposit a spot of the liquidsample on the substrate. The spot has a predetermined volume less thanthe volume of the droplet.

According to another aspect, a method of diluting a first liquid samplein a second liquid is provided. The method comprises providing adispensing system comprising two pins having a first tip and a secondtip separated from the first tip by a variable distance, acquiring adroplet of the first liquid sample between the pins and immersing thepin tips containing the droplet of the first liquid sample in a secondreservoir containing a second liquid, whereby the droplet of the firstliquid sample is diffused into the second liquid.

According to another aspect, a two-pin droplet dispensing system isprovided. The two-pin droplet dispensing system comprises a holder, afirst pin connected to the holder and having a first tip and a secondpin connected to the holder and having a movable tip spaced apredetermined distance from the first tip to form a sample acquisitionregion for holding a predetermined volume of liquid sample. The systemfurther comprises a driver for effecting movement of the movable tipwith respect to the first tip.

According to a final aspect, a liquid sample dispensing systemcomprising a holder a first pin having a first tip and a relaxationregion for absorbing an impact on the tip is provided.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 a and 1 b illustrate the two-pin dispensing system of anillustrative embodiment in a sample acquisition mode.

FIG. 2 illustrates the two-pin dispensing system of an illustrativeembodiment in a spotting mode.

FIGS. 3 a and 3 b illustrate the two-pin dispensing system of anillustrative embodiment in a dilution mode.

FIG. 4 illustrates an alternative embodiment of the two-pin dispensingsystem, including a fixed pin and a movable pin.

FIG. 5 is a detailed view of the fulcrum region of the two-pindispensing system of FIG. 4.

FIG. 6 is a detailed view of the driver of the two-pin dispensing systemof FIG. 4.

FIGS. 7 a, 7 b and 7 c are detailed views of the tip region of the twopins of the pin dispensing system of FIG. 4.

FIG. 8 illustrates the relaxation region of the pin dispensing system ofFIG. 4.

FIG. 9 is a scanning electron microscope (SEM) image of an array oftwo-pin dispensing systems that are microfabricated from a silicon waferaccording to the teachings of the illustrative embodiment of the presentinvention.

FIG. 10 is a SEM image showing a detailed view of a tip region of one ofthe two-pin dispensing systems of FIG. 9.

FIG. 11 is a SEM image illustrating an array of two-pin dispensingsystems having relaxation regions that are microfabricated from asilicon wafer according to the teachings of the illustrative embodimentof the present invention.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The present invention provides a dispensing system for dispensing apredetermined volume of liquid sample. The dispensing system of thepresent invention provides precise acquisition and delivery of a liquidsample to a sample handling system using two interacting pins. Thedispensing system is suitable for use in a basic research or acommercial environment. The dispensing system significantly improvessample introduction into a sample handling system by increasing theefficiency, speed and controllability of forming and dispensing dropletswhile significantly reducing waste and contamination. The invention willbe described below relative to an illustrative embodiment. Those skilledin the art will appreciate that the present invention may be implementedin a number of different applications and embodiments and is notspecifically limited in its application to the particular embodimentdepicted herein.

FIGS. 1 a and 1 b illustrate a two-pin dispensing system 10 of anillustrative embodiment of the present invention in a sample acquisitionmode for acquiring a droplet of a liquid sample having a predeterminedvolume from a reservoir containing a supply of the liquid sample. Theillustrative two-pin dispensing system 10 comprises a pair of separatelymovable, interacting pins sized and configured to hold a droplet ofliquid between the tips of the pins. The two-pin dispensing system 10comprises a first pin 11 and a second pin 12, which are movablyconnected to a holder 13. The tips 11 a, 12 a are separated by aninitial separation distance D to form a sample acquisition region 14 inthe space between the tips. The position of each pin is controlled usingactuators 15 located in one or more of the pins 11, 12. According to analternate embodiment, sensors 16 are provided on one or more of the pins11, 12 to measure the separation distance D between the pin tips 11 a,12 a. Those of ordinary skill in the art will readily recognize that theholder of the invention can include any suitable structure for retainingor holding the pins.

To acquire a droplet of a liquid sample, such as a biological sample,the pin tips 11 a, 12 a are immersed in a reservoir 17 containing asupply of a selected liquid sample. The pin tips 11 a, 12 a arepositioned to allow capillary flow into to sample acquisition region 14.The capillary force induced in the sample acquisition region 14 pulls adroplet 18, having a volume defined by the separation distance of thepin tips 11 a, 11 b, into the sample acquisition region 14. Thecapillary force produced between the surfaces of the pin tips holds thedroplet in the sample acquisition region 14 formed between the two pintips 11 a, 11 b. The actuators 15 in the pins 11, 12 move the pins tovary the separation distance D between the tips, thereby varying theamount of sample that is acquired by the two-pin dispensing system, orto dispense the sample therefrom.

According to the illustrative embodiment, the two-pin dispensing system10 is configured to acquire liquid samples in volumes between aboutfifty picoliters and about fifty nanoliters. One skilled in the art willrecognize that the acquired volume is not limited to this range and thatthe pins may be spaced apart to accommodate any suitable volume ofliquid.

The actuators 15 can also compensate for varying physical properties ofthe particular liquid sample, such as viscosity, surface tension, andthe like, by modifying the separation distance D between the pins. Thesensors 16 may also be utilized to measure the force applied between thetips and the physical properties of the acquired liquid sample on thefly. In this manner, the settings (i.e. the pin separation distance) ofthe pin dispensing system 10 can be modified to compensate forvariations in the measured properties of the liquid sample in real time.

According to the illustrative embodiment, the droplet dispensing system10 is fabricated from a silicon wafer using a microfabricationtechnique, such as a standard photolithography etching process, tofabricate the pin structures. One skilled in the art will recognize thatalternative materials and manufacturing techniques may be utilized. Forexample, the pin dispensing system may be made out of glass, plastic orany other suitable material. According to one embodiment, an array ofdroplet dispensing systems 10, each comprising two pins having avariable separation distance, may be formed on a single substrate, suchas a silicon wafer. For example, an array of up to about 300 or moretwo-pin dispensing systems 10 may be formed on a four-inch siliconwafer.

FIG. 2 illustrates the two-pin dispensing system 10 of the illustrativeembodiment in a spotting mode. The two-pin dispensing system 10 may beutilized as a spotting system for printing or discharging arrays ofbiochemicals, such as nucleic acid molecules or proteins, or othersuitable liquid samples to a sample handing system, such as a printingsubstrate, titre plate, microfluidic system or device, and the like foruse in proteomics, genomics, screening, diagnostics and otherapplications. After the dispensing system acquires a droplet, thedispensing system is moved in close proximity to a surface 20. Thesurface 20 may comprise a solid surface or a liquid. The surface 20 maycomprise a porous structure, such as a porous membrane, or a non-porousstructure, such as a microscope slide. The loaded pins deposit a spot 21on the surface 20 having a selected spot volume by direct contactbetween the pin tips 11 a, 11 b and the surface. The separation distanceD2 during contact may be varied to increase or decrease the volume ofthe dispensed spot of the liquid sample. According to the illustrativeembodiment, the volume of the dispensed spot 21 is significantly smallerthan the volume of the acquired droplet 18, and is generallysub-nanoliter in volume, though one skilled in the art will recognizethat the invention is not limited to this range.

The use of the two-pin dispensing system of the illustrative embodimentin spotting applications provided enhanced control over the size of thedeposited spots in a microarray, and also allows for smaller spots to beformed and deposited.

The pin dispensing system may further be utilized as a wet depositsystem with dilution to dilute a selected volume of a first liquid in asecond liquid sample. FIGS. 3 a and 3 b illustrate the two-pindispensing system 10 in a dilution mode, wherein the acquired droplet 18of a sample is diluted in a larger supply of a target fluid 30. Afterthe dispensing system 10 acquires a droplet 18, the size of which isdefined by the separation distance of the pin tips 11 a, 12 a, the pintips 11 a, 12 a are immersed in a reservoir 30 containing a targetfluid. The droplet 18 automatically dilutes into the target fluid viamixing and diffusion. To accelerate the dilution process, the separationdistance of the tips 11 a, 12 a may be increased during dilution usingthe actuators 15.

FIG. 4 illustrates a two-pin dispensing system 40 having a fixed pin anda movable pin according to an alternate embodiment of the invention. Inthe two-pin dispensing system of FIG. 4, the resting position of a firstpin 41 is fixed relative to a substrate 43 and the resting position ofthe second pin 42 is movable relative to the first pin 41 and thesubstrate 43. The two-pin dispensing system 40 further includes a driver44 for varying the separation distance between the tips 41 a, 42 a byadjusting the position of the second movable pin 42 in a fulcrum region46. According to the illustrative embodiment, the movable pin rotatesabout a fixed pivot point 45 under the control of the driver 44 toadjust the separation distance at the tips. According to theillustrative embodiment, the pins 41, 42 further include a relaxationregion 51 for preventing breakage of the tips. One skilled in the artwill recognize that the relaxation region 51 may be formed in one orboth of the pins 41, 42 of the two-pin dispensing system 40.

The illustrative two-pin dispensing system 40 is formed from a siliconwafer using a standard photolithography etching process to fabricate thepins 41, 42, the relaxation region 51, the driver 44, and the fixedpoint 45 of the fulcrum region 46 in the wafer substrate 43. Accordingto the illustrative embodiment, the two-pin dispensing system 40 isfabricated from a silicon wafer having dimensions of about one squarecentimeter. The pins 41,42 have a length of about five millimeters,though one skilled in the art will recognize that the invention is notlimited to this size. According to an alternate embodiment, a largersilicon wafer or other suitable substrate is provided, and an array ofpin dispensing systems is fabricated on the larger silicon wafer. Forexample, a silicon wafer having a size of about ten square centimetersmay be used to fabricate an array of about seventy two-pin dispensingsystems 40 thereon. A fifteen square centimeter silicon wafer can beutilized to fabricate over one hundred two-pin dispensing systems 40 inthe silicon wafer substrate. Those of ordinary skill will readilyrecognize that any suitable configuration can be employed to move one orboth of the pins.

FIG. 5 is a detailed view of the fulcrum region 46 of the two-pindispensing system of FIG. 4. The movable pin 42 is configured to pivotabout a fixed point 45 to vary the separation distance of the two pintips. The driver 44 applies a force to an application region 47 of thefulcrum region 46 to cause the movable pin 42 to rotate, therebyeffecting movement of the movable pin tip 42 a relative to the tip 41 aof the fixed pin 41. As illustrated, the fulcrum region 46 includes gaps48 are formed in the substrate 43 adjacent to the fixed point 45 toallow for rotation of the pin 42 about the fixed point in response toactivation of the driver 44.

According to an alternate embodiment of the invention, the fulcrumregion further includes bending sensors, illustrated as piezoresistors62, on the left and right side of the fulcrum region to allowdifferential sensing of actual bending of the pin 42 in the fulcrumregion. In this manner, the amount of bending, and the resultant tipseparation distance may be controlled using a closed loop feedbacksystem. The use of bending sensors further limits nonlinear temperatureeffects by allowing real-time sensing of tip displacement.

FIG. 6 is a detailed view of the driver 44 of the two-pin dispensingsystem 40 of FIG. 4. As shown the driver 44 comprises a bar of siliconthat imparts a force on the application region 47 of the fulcrum 46 tomove of tip 42 a a predetermined amount. According to the illustrativeembodiment, the driver 44 expands a predetermined amount in response toa temperature increase. The expansion of the driver 44 forces rotationof the fulcrum about the pivot point. According to the illustrativeembodiment, the system is configured such that the ratio between theamount of movement of the tip 42 a in response to expansion of thedriver 44 to the amount of expansion of the driver is greater than onehundred. In other words, a driver expansion of one micron causes a onehundred micron displacement of the pin tip 42 a.

According to the illustrative embodiment, the driver 44 has an initiallength L of four millimeters. A thirty-degree rise in temperature of thesilicon results in a 1.08 micrometer expansion of the driver 44. Theexpansion of the driver 44 forces the pin 42 to rotate about the fixedpivot point 45, thereby increasing the separation distance between thetips 41 a, 42 a by greater than 108 microns.

According to the illustrative embodiment, heating resistors 49 areaffixed to the driver for applying heat to the driver 44. The heatingresistors may comprise poly resistors, diffused resistors or anysuitable means for applying heat to the driver 44 in order to effectcontrolled expansion of the driver 44 and to vary the separationdistance between the tips 41 a, 42 a. Optionally, cooling fins (notshown) are provided in the driver 44 near the fulcrum region 45 toprevent unwanted heating of the driver in the fulcrum region. Accordingto an alternate embodiment, a temperature sensor 50 in communicationwith the heating means is included in the two-pin dispensing system 40to provide closed loop control of the driver 44 temperature.

One skilled in the art will recognize that the two-pin dispensing systemis not limited to the illustrative driver. According to alternateembodiments the driver 44 comprises an electrostatic system, apiezoelectric system, an electromechanical system, a thermoelectricactuator or any suitable system for applying a predetermined and definedforce to cause controlled adjustment of the separation distance betweenthe pin tips 41 a, 42 a. One skilled in the art will further recognizethat the two-pin dispensing system is not limited to a fulcrum forvarying the separation distance and that any suitable mechanism forvarying the separation distance may be utilized.

FIGS. 7 a and 7 b are detailed views of the tip region of the two pinsof the two-pin dispensing system 40 of FIG. 4. As discussed, the tips 41a, 42 a are spaced apart a predetermined distance D, which is defined bythe driver 44. Each tip includes a sample contact surface 70, defined bythe tip height H and the tip depth S, which are fixed values determinedby the shape of the sample surfaces 70. The volume of the sampleacquisition region 14 and thus the volume of an acquired sample dropletcorresponds to the volume of the space defined between the tips, or theproduct of the tip height H, the tip depth S and the separation distanceD. For example, according to the illustrative embodiment, the separationdistance D between the pin tips 41 a, 42 a is between about twenty-fivemicrons and about one hundred twenty five microns. For tips having a tipdepth of about fifty microns and a height of about two hundred microns,the resulting volume of a captured droplet is between about 250picoliters and about 1.25 nanoliters. For tips having a tip depth of 100microns and a height of about 400 microns, a tip separation distancebetween about 25 and 125 microns results in an acquired droplet having avolume of between about 1.0 nanoliters and about 5 nanoliters. Tipshaving a depth of 500 microns and a height of 500 microns, form adroplet having a volume between about 6.25 nanoliters and about 31.5nanoliters when the separation distance between the tips is betweenabout 25 and about 125 microns.

The tip contact surfaces 70 defined by the tip heights H and tip depthsS may form parallel faces or, according to a preferred embodiment, maybe tapered, so that the separation distance D is reduced towards thebottom and/or front of the tip surface. In this manner, smaller dropletvolumes may be accommodated. The slope of the tips 41 a, 42 a may bevaried in regions 71 and 72 to improve droplet shape and enhancedelivery of the droplet.

FIG. 7 c is a cross-sectional view of the tips 41 a, 42 a according toan alternate embodiment. According to the alternate embodiment, the tipsurfaces 70′ are curved to hold form a cylindrical or conical sampleacquisition region 14 therebetween.

According to alternate embodiment, the one or both of the tip surfaces70 and/or the outside shaft surface are coated with a hydrophilic,hydrophobic or other chemical coating to enhance droplet acquisition anddispensing. For example, the tips 41, 42 may be formed of or coated witha hydrophilic coating to enhance retention of a sample in the sampleacquisition region. According to one embodiment, the outside shaftsurfaces of the tips 41,42 are coated with gold or another suitablehydrophobic material without affecting the tip surfaces 70 defining thesample acquisition region 14. The use of a metal coating providesenhanced control over the volume and release of a droplet. The use ofsilicon and/or gold additionally allows for more vigorous cleaningsolutions to be utilized when cleaning the tips without degrading thesystem. In this manner, contamination of the tips is reduced.

The coating may be applied in a pattern to the tip surfaces 70 or theother surfaces of the tips 41, 42 by shadow masking. The coating may besputtered, or evaporated on a surface in a predetermined pattern,defined by a mask. One skilled in the art will recognize that anysuitable pattern for directing the liquid sample and enhancing controlover sample acquisition and dispensing may be utilized.

According to another embodiment, the dispensing system may comprise asingle pin having a suitable pattern coating applied to the surfaces ofthe pin tip. For example, the shaft of the single pin may be coated witha suitable hydrophobic coating and the tip of the pin may be coated witha suitable hydrophilic coating to enhance acquisition and dispensing ofa liquid sample.

FIG. 8 illustrates the relaxation region 51 of the pin dispensing system40 of FIG. 4. The pin tips 41, 42 are brittle and subject to breakagewhen accidentally touched down to surfaces, due to their size and thematerial used to fabricate the pins. The illustrative relaxation region51 comprises a spring 52 formed between the tip 41 a and the substrate43. When the tip 41 a contacts a surface, the spring absorbs the impulseand retracts the tip 41 a to prevent breakage. The springs 52 in thepins are configured to move the corresponding tip up and away from theother tip to prevent collision of the tips. The invention is not limitedto the illustrative spring design. One skilled in the art will recognizethat any suitable spring design may be utilized to form the relaxationregion 51 to protect the pin tips from breakage.

According to an alternate embodiment, the spring 52 includes sensors tomeasure of the force of contact between the tip and a surface. Forexample, differential piezoresistive sensors may be included in thespring 52 and connected to an actuator (not shown) to control the springusing feedback control loop. The spring sensor may also be utilized tomeasure the force exerted by the droplet on the tips, and allow thedriver to compensate for variable forces exerted by the droplet on thetips.

According to an alternate embodiment of the present invention, arelaxation region may be implemented in a two-pin dispensing systemcomprising a pair of spaced-apart, fixed pins defining a sampleacquisition region of fixed volume.

As discussed, the two-pin dispensing system 10 or 40 of the illustrativeembodiment may be microfabricated from a suitable substrate, such assilicon, glass or plastic. According to the illustrative embodiment,photolithography may be utilized to form the pin structures in thesubstrate. In photolithography, the pattern of the two pins and othercomponents of the two-pin dispensing system 10 or 40 are imprinted on asilicon wafer, or other substrate, using one or more photoresist layersthat are patterned by UV or other light projected through one or morephoto-masks containing the pattern on it. The substrate is then etchedto fabricate the two-pin structure. One skilled in the art willrecognize that any suitable microfabrication technique may be utilizedto manufacture the two-pin dispensing system of the illustrativeembodiment of the present invention.

One skilled in the art will recognize that the describedmicrofabrication technique may further be utilized to fabricatesingle-pin dispensing systems from a silicon wafer or other suitablesubstrate. For example, it is within the scope of the invention tomicrofabricate a single-pin structure having two tips forming a samplechannel for acquiring and dispensing a liquid sample, as described inU.S. Pat. No. 6,101,946, from a silicon wafer by etching the siliconwafer to define the pin and sample channel.

FIG. 9 is a scanning electron microscope (SEM) image of an array oftwo-pin sample dispensing systems 100 according to an embodiment of theinvention and formed from a silicon wafer 101 using the above-describedmicrofabrication technique. As shown, a plurality of two-pin dispensingsystems are fabricated from a single silicon wafer substrate. Eachtwo-pin dispensing system comprises a pair of elongated pins that arespaced apart to define a sample acquisition region between the tips ofthe pins.

FIG. 10 is another SEM image showing a detailed view of the tip regionof one of the microfabricated two-pin dispensing systems of FIG. 9. Asshown, the pins are etched in a silicon wafer to define a sampleacquisition region 140 between the tips of the pins. As illustrated, themicrofabricated pin tips have a separation distance of less than about100 microns.

FIG. 11 is another SEM image of an array of microfabricated two-pinsample dispensing systems 110 having relaxation regions 51 according toan embodiment of the invention. As shown, the array is also formed froma silicon wafer 101 using the above-described microfabricationtechnique. The relaxation region 51 is formed by etching the siliconwafer in the region between the pin tips and the holder to define aspring for absorbing an impact on the tips. The relaxation region 51prevents breakage of the pin tips 41, 42 when the pin tips contact asurface.

The two-pin dispensing system provides significant improvements to theprocess of forming and dispensing droplets of samples for spotting anddilution applications. The illustrative configuration provides precisecontrol over the amount of liquid sample that is acquired and depositedthrough the use of two pins having a variable separation distance.Adjusting the separation distance between the pin tips easily andprecisely modifies the volume of the acquired liquid droplet and thedeposited liquid droplet. Furthermore, measurements of the physicalproperties of the liquid volume can be made on the fly and the tipseparation can be modified quickly and easily to compensate forvariations. The use of sensors provides precise control of the tipseparation distance to optimize the process of acquiring and dispensingdroplets of a liquid sample.

The present invention has been described relative to an illustrativeembodiment. Since certain changes may be made in the above constructionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description or shown in theaccompanying drawings be interpreted as illustrative and not in alimiting sense.

It is also to be understood that the following claims are to cover allgeneric and specific features of the invention described herein, and allstatements of the scope of the invention which, as a matter of language,might be said to fall therebetween.

1. A method of fabricating a liquid sample dispensing system,comprising: providing a substrate; applying a pattern to the substrateto define a holder, a first pin having a first tip and a second pinhaving a second tip spaced from the first tip to define a sampleacquisition region; and etching the substrate to remove excess portionsof the substrate, thereby fabricating the liquid sample dispensingsystem.
 2. The method of claim 1, wherein the substrate comprises asilicon wafer.
 3. The method of claim 1, wherein the substrate comprisesa glass plate.
 4. The method of claim 1, further comprising the step ofapplying a chemical coating to at least one of the first pin and thesecond pin.
 5. The method of claim 1, wherein the first and second pinsare movable relative to each other.
 6. The method of claim 1, furthercomprising the step of coupling an actuator to the etched substrate forvarying a separation distance between the first tip and the second tip.7. The method of claim 1, further comprising the step of coupling asensor to the etched substrate for sensing the separation distancebetween the first tip and the second tip.
 8. The method of claim 1,further comprising the step of coupling a sensor to the etched substratefor measuring the physical properties of a liquid sample held in thesample acquisition region.
 9. The method of claim 8, further comprisingthe step of coupling an actuator to the etched substrate, wherein theactuator varies the separation distance in response to the measurementof the physical properties.
 10. The method of claim 1, wherein thepattern includes a driver for moving the first pin relative to thesecond pin.
 11. The method of claim 1, wherein the first pin and thesecond pin have a length of about five millimeters.
 12. The method ofclaim 1, wherein the sample acquisition region has a volume betweenabout 6.25 nanoliters and about 31.5 nanoliters.
 13. The method of claim1, wherein an array of liquid sample dispensing systems is formed on asingle substrate.
 14. A method of microfabricating a liquid sampledispensing system, comprising the steps of: providing a silicon wafer;applying a pattern to the silicon wafer to define a holder, a first pinhaving a first tip and a second pin having a second tip spaced from thefirst tip to define a sample acquisition region; and etching the siliconwafer to remove excess portions of the substrate, thereby fabricatingthe liquid sample dispensing system.
 15. The method of claim 1, whereinthe silicon wafer has dimensions of about one square centimeter.
 16. Themethod of claim 1, wherein an array of liquid sample dispensing systemsis formed on a single silicon wafer.
 17. A method of fabricating aliquid sample dispensing system, comprising: providing a substrate;applying a pattern to the substrate to define a holder and a first pinhaving a first tip defining a sample acquisition region; etching thesubstrate to remove excess portions of the substrate, therebyfabricating the liquid sample dispensing system; and applying a chemicalcoating to the first pin.
 18. The method of claim 17, wherein thechemical coating comprises one of a hydrophobic material and ahydrophilic material.
 19. The method of claim 17, wherein the chemicalcoating is applied in a predetermined pattern determined by a mask usedin applying the chemical coating to the tip.
 20. The method of claim 17,wherein pattern also defines a second tip that is etched in thesubstrate spaced from the first tip to define the sample acquisitionregion.